Part:BBa_J52008

luciferase: luciferin 2-monooxygenase from Renilla reniformis (EC 1.13.12.5; SwissProt:: P27652)

Rluc is a protein called Renilla luciferase and it emits light when the right substrate is added. That is why it can be used as a reporter to track other proteins or to monitor the activity of a promoter.

Usage and Biology

This part contains a Eukaryotic ribosome binding site and non-standard prefix and suffix. See the sequence first.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI.rc site found at 547
  

This part was characterized by the iGEM team DUT_CHINA_B in 2019.

1.Introduction

Renilla-luciferin 2-monooxygenase, Renilla luciferase, or RLuc, is a bioluminescentenzyme found in Renilla reniformis, belonging to a group of coelenterazine luciferases. Of this group of enzymes, the luciferase from Renilla reniformis has been the most extensively studied, and due to its bioluminescence requiring only molecular oxygen, has a wide range of applications, with uses as a reporter gene probe in cell culture, in vivo imaging, and various other areas of biological research[1].
RLuc catalyzes the chemical reaction ：
Coelenterazine + O2→coelenteramide + CO2 + hν
In the process, coelenterazine is oxidized with a concurrent loss of CO2, and a photon of blue light is emitted[2].

2. Characterization

For the configuration, preservation and use of coelenterin solution, see protocol Absorption measurement of Fluorescence.pdf
Determination of optimal incubation time of Rluc.
After the 3ml system is configured, 2900ul crude enzyme solution and 100mul coelenterin working solution should be added, and then measure the luminescence intensity at 480nm after incubation for 0,5,10,15,20,25,30min.
Determination of optimal substrate concentration of Rluc.
The system was configured to 3ml, and an appropriate amount of crude enzyme solution and the prepared coelenterin working solution should be added to make its working concentration 0,5,10,15,20 uM respectively. After incubation for 10min, measure the luminescence intensity at 480nm .

3.Result and Discussion

As can be seen from the figure 1 and table 1, the relative luminescence intensity increased with the increase of incubation time, and the growth rate was faster in the first 10 minutes.

As can be seen from the figure 2 and table 2, the maximum luminescence intensity appeared at the substrate of 10 uM, so the subsequent experimental coelenterin substrate concentration was determined to be 10 uM.

4.references

[1]Daunert S, Deo S K. Photoproteins in Bioanalysis[J]. Wiley, 2006. [2]Schomburg D, Salzmann M. Enzyme Handbook[J]. 2001.